We have determined that (i) pH-dependent cleavage of the glycosyl phosphatidylinositol (GPI) anchored protein GP2 from the ectoleaflet of the apical plasma membrane of rat pancreatic acinar cells plays a critical role in the activation of endocytosis; II this endocytic process is mediated through activation of tyrosine kinases; and (iii) GP2 appears to activate this process through the formation of a complex with the src kinases pp60 and p62yes as well as caveolin. These data indicate that ductal bicarbonate secretion, through alkalinization of the acinar lumen, regulates apical endocytosis in acinar cells through the pH-dependent cleavage of GP2. In support of this hypothesis we have shown in Cystic Fibrosis (CF) knockout mice that the absence of bicarbonate and hence the lack of alkalinization of the acinar lumen leads to a selective impairment in GP2 release as well as apical endocytosis. We have also discovered that CF knockout mice have a specific fatty acid imbalance characterized by an increase in phospholipid-bound arachidonic acid (AA) and a decrease in phospholipid-bound docosahexaenoic acid (DHA). This was present only in CFTR regulated tissues. Correction of this fatty acid imbalance by oral administration of DHA to CF mice resulted in reversal of the pathology in the pancreas, ileum, and in pseudomonas endotoxin-induced lung inflammation. In addition, we have recently shown that humans with CF have a similar abnormality in AA and OHA. The underlying hypothesis for this proposal is that the imbalance in AA and DHA may (i) lead to the excessive inflammatory response characteristic of CF and (ii) play a role in apical membrane events in the exocrine pancreas. This proposal will build upon these observations and (i) determine the mechanism by which defects in CFTR lead to abnormalities in AA and DHA; (ii) determine the role of fatty acids in membrane events at the apical surface of acinar cells; and (iii) determine the mechanism by which DHA regulates inflammation. Results from this project will be important in understanding the pathophysiology of CF and the development of potential therapies for diseases related to CF gene mutations.